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Dealing with Resolution Anisotropy andConsiderations for High Resolution Structure
Determination
Dmitry LyumkisSalk Institute for Biological Studies
American Crystallographic Association, cryo-EM Workshop, August 11, 2020
• Preferred orientation — the origin of resolution anisotropy
Outline
• Resolution anisotropy in cryo-EM reconstructions (caused
by preferred orientation) and its evaluation with 3D FSCs
• Combatting anisotropy using tilted data collection strategies
• The (unexpected) effect of sampling (in)homogeneity on
global resolution
• Evaluating sampling (in)homogeneity using the SCF criterion
• Considerations for high-resolution
Origin of resolution anisotropy in cryo-EM:The idealized behavior of particles on a grid
Noble, A. J., et al. (2018). Routine single particle CryoEM sample and grid characterization bytomography. eLife, 7, 32.
Noble, A. J., et al. (2018). Routine single particle CryoEM sample and grid characterization bytomography. eLife, 7, 32.
Origin of resolution anisotropy in cryo-EM:The reality … Particles adhere at the air-water interface
Origin of resolution anisotropy in cryo-EM:The reality … for virtually all samples
Noble, A. J., et al. (2018). Routine single particle CryoEM sample and grid characterization bytomography. eLife, 7, 32.
• Preferred orientation — the origin of resolution anisotropy
Outline
• Resolution anisotropy in cryo-EM reconstructions (caused
by preferred orientation) and its evaluation with 3D FSCs
• Combatting anisotropy using tilted data collection strategies
• The (unexpected) effect of sampling (in)homogeneity on
global resolution
• Evaluating sampling (in)homogeneity using the SCF criterion
• Considerations for high-resolution
90°
Interaction at the air-water interface results in:“preferred particle orientation” — Hemagglutinin (HA) trimer
Tan, Y. Z. et al. (2017). Nature Methods, 14(8), 793–796.
Interaction at the air-water interface results in:smearing and elongation along Z-axis (loss of Z resolution)
Tan, Y. Z. et al. (2017). Nature Methods, 14(8), 793–796.
beamdirection
beamdirection
Fou
rier
Tra
nsf
orm
Hal
f Map
1H
alf M
ap 2
Spatial Frequency
Four
ier
Shel
lC
orre
lati
on
Fourier Shell Correlation (FSC): a quantitative way toevaluate resolution in cryo-EM
● Calculate the FSC between two half maps as afunction of spatial frequency over the entireFourier shell
Harauz, G., & van Heel, M. (1986). Exact filters for general geometry 3-dimensionalreconstruction. Optik, 73(146), 146–156.
Fou
rier
Tra
nsf
orm
Hal
f Map
1H
alf M
ap 2
Spatial Frequency
Four
ier
Shel
lC
orre
lati
on
● Calculate the FSC between two half maps as afunction of spatial frequency over the entireFourier shell
Harauz, G., & van Heel, M. (1986). Exact filters for general geometry 3-dimensionalreconstruction. Optik, 73(146), 146–156.
Fourier Shell Correlation (FSC): a quantitative way toevaluate resolution in cryo-EM
Fou
rier
Tra
nsf
orm
Hal
f Map
1H
alf M
ap 2
Spatial Frequency
Four
ier
Shel
lC
orre
lati
on
● Calculate the FSC between two half maps as afunction of spatial frequency over the entireFourier shell
Harauz, G., & van Heel, M. (1986). Exact filters for general geometry 3-dimensionalreconstruction. Optik, 73(146), 146–156.
Fourier Shell Correlation (FSC): a quantitative way toevaluate resolution in cryo-EM
Fou
rier
Tra
nsf
orm
Hal
f Map
1H
alf M
ap 2
Spatial Frequency
Four
ier
Shel
lC
orre
lati
on
● Calculate the FSC between two half maps as afunction of spatial frequency over the entireFourier shell
Harauz, G., & van Heel, M. (1986). Exact filters for general geometry 3-dimensionalreconstruction. Optik, 73(146), 146–156.
Fourier Shell Correlation (FSC): a quantitative way toevaluate resolution in cryo-EM
Fou
rier
Tra
nsf
orm
Hal
f Map
1H
alf M
ap 2
Spatial Frequency
Four
ier
Shel
lC
orre
lati
on
● Calculate the FSC between two half maps as afunction of spatial frequency over the entireFourier shell
Harauz, G., & van Heel, M. (1986). Exact filters for general geometry 3-dimensionalreconstruction. Optik, 73(146), 146–156.
Fourier Shell Correlation (FSC): a quantitative way toevaluate resolution in cryo-EM
● Calculate the FSC between two half maps as afunction of spatial frequency over the entireFourier shell
Fou
rier
Tra
nsf
orm
Hal
f Map
1H
alf M
ap 2
Spatial Frequency
Four
ier
Shel
lC
orre
lati
on
Harauz, G., & van Heel, M. (1986). Exact filters for general geometry 3-dimensionalreconstruction. Optik, 73(146), 146–156.
Fourier Shell Correlation (FSC): a quantitative way toevaluate resolution in cryo-EM
● Calculate the FSC between two half maps as afunction of spatial frequency over the entireFourier shell
Fou
rier
Tra
nsf
orm
Hal
f Map
1H
alf M
ap 2
Spatial Frequency
Four
ier
Shel
lC
orre
lati
on
Harauz, G., & van Heel, M. (1986). Exact filters for general geometry 3-dimensionalreconstruction. Optik, 73(146), 146–156.
FSC 0.143
Fourier Shell Correlation (FSC): a quantitative way toevaluate resolution in cryo-EM
● Calculate the FSC between two half maps as afunction of the direction (and looking atcross-correlations over a local cone of dataand not the entire shell)
3D FSCs: a quantitative way to evaluate directional resolutionand anisotropy in single-particle cryo-EM
Tan, Y. Z. …, Lyumkis D. (2017). Nature Methods, 14(8), 793–796.
● Calculate the FSC between two half maps as afunction of the direction (and looking atcross-correlations over a local cone of dataand not the entire shell)
Fou
rier
Tra
nsf
orm
Hal
f Map
1H
alf M
ap 2
Spatial Frequency
Four
ier
Shel
lC
orre
lati
on
Tan, Y. Z. …, Lyumkis D. (2017). Nature Methods, 14(8), 793–796.
3D FSCs: a quantitative way to evaluate directional resolutionand anisotropy in single-particle cryo-EM
● Calculate the FSC between two half maps as afunction of the direction (and looking atcross-correlations over a local cone of dataand not the entire shell)
Fou
rier
Tra
nsf
orm
Hal
f Map
1H
alf M
ap 2
Spatial Frequency
Four
ier
Shel
lC
orre
lati
on
FSC at View #1
Tan, Y. Z. …, Lyumkis D. (2017). Nature Methods, 14(8), 793–796.
3D FSCs: a quantitative way to evaluate directional resolutionand anisotropy in single-particle cryo-EM
● Calculate the FSC between two half maps as afunction of the direction (and looking atcross-correlations over a local cone of dataand not the entire shell)
Fou
rier
Tra
nsf
orm
Hal
f Map
1H
alf M
ap 2
Spatial Frequency
Four
ier
Shel
lC
orre
lati
on
FSC at View #2
Tan, Y. Z. …, Lyumkis D. (2017). Nature Methods, 14(8), 793–796.
3D FSCs: a quantitative way to evaluate directional resolutionand anisotropy in single-particle cryo-EM
● Calculate the FSC between two half maps as afunction of the direction (and looking atcross-correlations over a local cone of dataand not the entire shell)
Fou
rier
Tra
nsf
orm
Hal
f Map
1H
alf M
ap 2
Spatial Frequency
Four
ier
Shel
lC
orre
lati
on
FSC at View #3
Tan, Y. Z. …, Lyumkis D. (2017). Nature Methods, 14(8), 793–796.
3D FSCs: a quantitative way to evaluate directional resolutionand anisotropy in single-particle cryo-EM
● Calculate the FSC between two half maps as afunction of the direction (and looking atcross-correlations over a local cone of dataand not the entire shell)
Fou
rier
Tra
nsf
orm
Hal
f Map
1H
alf M
ap 2
Conical FSCsAcross Entire
Sphere
Tan, Y. Z. …, Lyumkis D. (2017). Nature Methods, 14(8), 793–796.
3D FSCs: a quantitative way to evaluate directional resolutionand anisotropy in single-particle cryo-EM
● Calculate the FSC between two half maps as afunction of the direction (and looking atcross-correlations over a local cone of dataand not the entire shell)
Fou
rier
Tra
nsf
orm
Hal
f Map
1H
alf M
ap 2
Conical FSCsAcross Entire
Sphere
3D FSC
Tan, Y. Z. et al. (2017). Nature Methods, 14(8), 793–796.
3D FSCs: a quantitative way to evaluate directional resolutionand anisotropy in single-particle cryo-EM
Evaluating resolution anisotropy: the 3D FSC
Tan et al. (2017). Nat Methods, 14(8), 793-796. Aiyer et al. in press. MiMB.
beamdirectio
n
beamdirectio
n
beamdirectio
n
Anis
otro
pic
case
Isot
ropi
c ca
se
Implementation of 3D FSCs in Chimera
Aiyer et al. in press. MiMB.
Web-server for remote 3D FSC processing:https://3dfsc.salk.edu
www.github.com/LyumkisLab/3DFSC
http://www.github.com/LyumkisLab/3DFSC
• Preferred orientation — the origin of resolution anisotropy
Outline
• Resolution anisotropy in cryo-EM reconstructions (caused
by preferred orientation) and its evaluation with 3D FSCs
• Combatting anisotropy using tilted data collection strategies
• The (unexpected) effect of sampling (in)homogeneity on
global resolution
• Evaluating sampling (in)homogeneity using the SCF criterion
• Considerations for high-resolution
Tilting the grid in the microscope helps to alleviateresolution anisotropy caused by preferred specimen
orientation
Lyumkis, D. (2019). JBC, 294(13), 5181–5197.
Tan et al. (2017). Nat Methods, 14(8), 793-796.
Why do tilts work?Filling in more Fourier space density (better Fourier sampling)
reconstruct
reconstruct
Tilting ameliorates resolution anisotropy and improves maps
Unt
ilted
Tilte
d
Anisotropic reconstructionuntilted data
(more) isotropic reconstructiontilted data
Passos, Li et al. (2020). Science
Tilting ameliorates resolution anisotropy and improves mapsHIV-1 intasome complexes (from Thursday ACA session)
Examples of tilted data collection strategies in the literature
Zhang, Y., … Shi, Y. (2020). bioRxiv, 9,2020.03.27.009381
~100 MDa Nuclear Pore Complex
Herzik, M. A., Wu, M., & Lander, G. C. (2019).Nature Communications, 10(1), 1–9.
~43 kDa Protein Kinase A
Possible limitations associated with tilted datacollection strategies
1. Defocus gradient across the micrograph
2. More beam-induced movement
3. Thicker ice
Compensating for limitations of tilts usingcomputational image processing: AAV
Working hypothesis: most of the problems associated with tilts couldbe ameliorated using computational strategies
0°st
age
tilt
60°s
tage
tilt
• Preferred orientation — the origin of resolution anisotropy
Outline
• Resolution anisotropy in cryo-EM reconstructions (caused
by preferred orientation) and its evaluation with 3D FSCs
• Combatting anisotropy using tilted data collection strategies
• The (unexpected) effect of sampling (in)homogeneity on
global resolution
• Evaluating sampling (in)homogeneity using the SCF criterion
• Considerations for high-resolution
75K particles
Passos, D. O., & Lyumkis, D. (2015). Journal of Structural Biology, 192(2), 235–244.
High-resolution dataset, with multiple “preferredorientations” for characterizing the effects of sampling
75K particles 10K particles
10K particlesorientation 1
10K particlesorientation 3
10K particlesorientation 2
Dataset could be subdivided into four groups by orientation
Reconstructions differing only in orientation distributionshave distinct SSNR profiles
• Preferred orientation — the origin of resolution anisotropy
Outline
• Resolution anisotropy in cryo-EM reconstructions (caused
by preferred orientation) and its evaluation with 3D FSCs
• Combatting anisotropy using tilted data collection strategies
• The (unexpected) effect of sampling (in)homogeneity on
global resolution
• Evaluating sampling (in)homogeneity using the SCF criterion
• Considerations for high-resolution
Dataset signal power /noise power at different
spatial frequencies
Average numberof times lattice
point k is sampled
Geometricalfactor
Per-particle SSNR
Heymann, J. B., IUCr. (2019). Single-particle reconstruction statistics: a diagnostic tool in solving biomolecularstructures by cryo-EM. Acta Crystallographica. Section F, Structural Biology and Crystallization Communications,
75(1), 33–44.
Decoupling sampling with noise variance
Derivation decouples a sampling factor from the per-particle SSNR
Previously …
Baldwin, P. R., & Lyumkis, D. (2019). Progress in Biophysics and Molecular Biology.http://doi.org/10.1016/j.pbiomolbio.2019.09.002
Evaluating SCF behavior using different Fourier samplingschemes
Decrement of the SCF for side-like views: (fully sampled)
Decrement of the SCF for top-like views:(Incompletely sampled)
45-degree cone,10% sprinkles
45-degree cone,3% sprinkles
45-degree cone,1% sprinkles
45-degree cone,0% sprinkles
Uniform
Knowing the SCF can predict the attenuation of the SSNR:side-like distributions, varying phi-angle modulation
Knowing the SCF can predict the attenuation of the SSNR:side-like distributions, varying phi-angle modulation
Knowing the SCF can predict the attenuation of the SSNR:top-like distributions, varying cone size with 3% “sprinkles”
Knowing the SCF can predict the attenuation of the SSNR:top-like distributions, varying cone size with 3% “sprinkles”
GUI for calculating SCF values:Example with top-like distribution, untilted and tilted
Baldwin, P. R., & Lyumkis, D. (2020).bioRxiv, 3(1), 2020.06.08.140863.
www.github.com/LyumkisLab/SamplingGui
Baldwin, P. R., & Lyumkis, D. (2020).PBMB, Jul 6:S0079-6107(20)30060-2
SCF* = 0.02
SCF* = 0.49
http://www.github.com/LyumkisLab/SamplingGui
• Preferred orientation — the origin of resolution anisotropy
Outline
• Resolution anisotropy in cryo-EM reconstructions (caused
by preferred orientation) and its evaluation with 3D FSCs
• Combatting anisotropy using tilted data collection strategies
• The (unexpected) effect of sampling (in)homogeneity on
global resolution
• Evaluating sampling (in)homogeneity using the SCF criterion
• Considerations for high-resolution
Considerations for optimizing cryo-EM resolution• Microscope (e.g. JEOL, TFS, Krios G3/G4, Arctica, etc) microscope settings (e.g. KV, aperture
size), microscope alignment (coma-free, parallel illumination)• Ancillary hardware: cold or warm FEG, energy filter, aberration correctors, phase plates• Detector and detector DQE: Falcon4, Falcon3, K3, K2, CCD, etc• Detector settings: counting vs. super-resolution, dose rate, frame rate• Imaging and data collection: magnification (and pixel size), defocus range, image shift or stage
position, speed vs accuracy (without compromising quality), hole selection, with or w/o stage tilt
• Grid / data collection quality: thick/thin ice, how much drift, sample dispersity, substrate support,grid type
• Image processing:◦ Software (Relion, CryoSparc, CisTEM, Warp/M, EMAN, etc)◦ Initial motion correction and CTF estimation (global, patch-based, per-particle)◦ Classification strategy◦ Post-processing (CTF refinement, particle polishing [movement, B-factor weighting], Ewald
correction, density modification)
• Fourier space sampling• Sample !!!!!!!!!!!!!!!!!!!!!!!!!
Nakane, T., …, Scheres, S. (2020). Single-particle cryo-EM at atomic resolution.bioRxiv, 2020.05.22.110189.
Considerations for optimizing cryo-EM resolution:1.2 Å apoferritin (cold FEG)
Yip, K. M., Fischer, N., Paknia, E., Chari, A., & Stark, H. (2020). Breaking the next Cryo-EMresolution barrier – Atomic resolution determination of proteins! bioRxiv, 645,
2020.05.21.106740.
Considerations for optimizing cryo-EM resolution:1.2 Å apoferritin (warm FEG)
• Titan Krios (Schottky FEG)• Voltage: 300• Monochromator + Cs corrector• Falcon 3 detector• 0.492 Å/pix• Electron dose (e-/Å^2): 50• Software: Relion• Number of particles: 1,090,676• Resolution: 1.25 Å
Considerations for optimizing cryo-EM resolution:1.3 Å apoferritin (cold FEG)
Tegunov, D., Xue, L., Dienemann, C., Cramer, P., & Mahamid, J. (2020). Multi-particle cryo-EM refinement with Mvisualizes ribosome-antibiotic complex at 3.7 Å inside cells. bioRxiv, 44, 2020.06.05.136341.
• cryoARM300 (JEOL)• Voltage: 300• Cold FEG• K2 detector• In-column omega filter• 0.49 Å/pix• Electron dose (e-/Å^2): 88• Software: M• Number of particles: 120,295• Resolution: 1.34 Å
Dimitry TegunovReprocessed dataset in M
EMPIAR-10248
Keiichi NambaData collection (Kato et al, MIcroscopy
and Microanalysis, 2019)
Rado DanevYanagisawa-san, Masahide
Kikkawa
Considerations for optimizing cryo-EM resolution:1.3 Å apoferritin (warm FEG)
Try to understand your limitationsFirst 2 e-/Å2
Tan, Y. Z., Aiyer, S., et al. (2018). Nature Communications, 9(1), 3628–11.
Exposure Dependency of the Mean Intensityof the Unmerged Integrated Reflections
Hattne, J., Shi, D., et al. (2018). Analysis of Global and Site-Specific Radiation Damage inCryo-EM, 26(5), 759–766.e4.
• Resolution anisotropy originates from preferred orientation and
inhomogeneity in Fourier sampling
• 3D FSCs provide quantitative measurements of resolution
anisotropy
• Tilts help to overcome resolution anisotropy
• Inhomogeneity in Fourier sampling also causes attenuation of
global resolution: incomplete sampling worse than
inhomogeneous (but complete) sampling
• The Sampling Compensation Factor (SCF) estimates the
amount of global resolution attenuation
• Many factors to optimize for high-resolution … toward sub-Å
reconstructions!
Summary
Acknowledgements and Support
Computationalsupport
Phil BaldwinJC Ducom
Funding
Collaborators
Niko GrigorieffBen Himes
Mavis Agbanjie-McKenna
Robert McKenna
Cryo-EM supportBill Anderson
Scripps/SalkConsortium
Jamie WilliamsonTravis BerggrenMartin Hetzer
Leona M. and Harry B. HelmsleyCharitable Trust Grant
Phil BaldwinYong Zi Tan Sriram Aiyer